口腔扁平苔藓相关lncRNA的鉴定及其对Th17细胞增殖和功能的影响

Oral lichen planus (OLP) is commonly considered as an immune oral mucosal disease induced by T lymphocytes. The pathogenesis still remains unknown. It's now confirmed that a new type of T cell subset, Th17 cells have taken part in many autoimmune diseases and have an important impact on OLP. The applicant had investigated that the functional molecules of the Th17 cells like IL-17 and IL-22 had abnormal expression which is in accordance with latest reported results of the LP patients' serum. The expression profile of long non-coding RNAs (lncRNAs) in human oral mucosa had been recently founded. And it had been confirmed that the expression of lncRNAs is crucial to the oral mucosal transcriptome. But there has been no report on the research of the expression profile of lncRNAs or the target gene in OLP tissue. Therefore, the applicant plans to conceive the lncRNAs expression profile in OLP and select several lncRNAs serve as biological tags to investigate the impact of the lncRNAs and their target molecules on the Th17 cells' proliferation, and the biological functional effect. The expression profile of lncRNAs of OLP tissue is expected. It's also anticipated that the mechanism of the lncRNAs' regulation on Th17 cells and the relation between the lncRNAs and OLP, which could be a basis for lncRNAs in biological treatment on OLP.

口腔扁平苔藓（OLP）是一种T淋巴细胞介导的免疫性口腔黏膜病，发病机制尚不明确。现已证实新型的T细胞亚群- - Th17细胞参与了多种自身免疫性疾病的发生发展。申请人前期发现Th17细胞功能性分子IL-17和IL-22在OLP病损中表达异常，与LP患者血清学研究报道一致。提示Th17细胞极其可能参与了OLP的发生，但其机制未明。最近有研究者构建了人类口腔黏膜lncRNA表达谱，证实lncRNA的表达对口腔黏膜转录组具有重要意义。目前尚无OLP组织中lncRNA的表达谱与功能的研究报道。因此申请人拟构建OLP的lncRNA表达谱，筛选可作为生物标记物的lncRNA，探讨lncRNA及其靶分子对Th17细胞增殖和功能的作用以及对OLP的影响。预期可建立OLP组织lncRNA的表达谱，初步明确lncRNA调控Th17细胞的机制及其与OLP发病的联系，为今后以lncRNA作为OLP治疗新靶点提供依据。

研究背景：OLP是一种T淋巴细胞介导的口腔黏膜病，发病机制尚不明确。Th17细胞可能参与了OLP的发生，但其机制未明。有研究者构建了人类口腔黏膜lncRNA表达谱，证实lncRNA对口腔黏膜转录组具有重要意义。目前尚无OLP组织lncRNA表达谱的研究报道。本课题组拟构建OLP的lncRNA表达谱，筛选可作为生物标记物的lncRNA，探讨lncRNA及其靶分子对Th17细胞的作用以及对OLP的影响。.研究内容：1.流式细胞术检测OLP患者外周血Th17细胞。2.构建OLP患者的lncRNA表达谱。3.筛选Th17细胞相关lncRNA并预测期靶基因，构建慢病毒载体，侵染HaCaT细胞，采用Real-time PCR和Western blot检测lncRNA和靶基因的表达。双荧光素酶报告法验证lncRNA与靶基因是否结合。.研究结果：1.OLP患者Th17细胞在CD4+ T细胞中的百分率为：(1.947±0.737)%；对照组为：(2.236±0.950)%，两者没有统计学差异（P＞0.05）。2.芯片结果发现OLP组织lncRNA谱中2倍以上变化的共180条，其中升高的共51条，降低的共129条。5倍以上变化的共21条，其中升高的共10条，降低的共11条。3.通过生物信息学方法分析，180条2倍以上变化的lncRNA中未发现直接调控IL-17A或IL-22的lncRNA。其中上调倍数最大（48.08倍）的ENST00000433410经预测靶基因为CXCR3。慢病毒侵染后，shRNA组、过表达组、空白对照组的ENST00000433410相对表达量分别为0.1772±0.0153、3.4130±0.0401、1.000；三组的CXCR3相对表达量分别为0.5756±0.0383、2.164±0.0679、1.000。Western Blot结果显示shRNA组CXCR3表达降低，过表达组CXCR3表达增高。双荧光素酶法证实ENST00000433410能够特异性结合CXCR3。.研究意义：OLP组织lncRNA表达谱的构建成功，为深入研究lncRNA在OLP发病中的作用提供了基础。首次发现ENST00000433410可能通过影响CXCR3的表达，间接调控Th17细胞的趋化，参与OLP的发生发展，为OLP的生物治疗提供新靶点，为研究OLP的发病机制提供新的思路和方法。